Stimulated secretion of granules from cultured RBL-2H3 mast cells is dependent on mobilization of calcium via phospholipase (PL) C and activation of protein kinase C by diglycerides generated through PLC and PLD. In recent years we have focussed on PLD because this enzyme is the primary source of diglycerides in stimulated mast cells and its activation is essential for secretion (see previous reports in this series). Stimulation of PLD is enhanced markedly in cells made to overexpress beta/gamma subunits of trimeric G proteins, by provision of recombinant beta/gamma subunits to permeabilized cells, and by treatment of cells with cholera toxin. We have hypothesized that G protein beta/gamma subunits interact with the recently identified pleckstrin homology (PH) domain in PLD to synergize other stimulatory signals. Studies in permeabilized cells and membrane preparations indicate that other signals may include physiologic increases in free ionized calcium, possibly by activating CAM kinase II, and activation of protein kinase C. Although RBL-2H3 cells contain the Arf-dependent PLD1 and the Arf-independent PLD2 isoforms of PLD, the latter isoform appears to account for the phenomena described above in that a cholera toxin-sensitive, Arf-independent PLD is located largely in the plasma membrane and, to a lesser extent, granule membrane (as determined by subcellular fractionation) along with PLD2. The membrane PLD can be stimulated directly in membranes with G protein stimulants such as mastoparan. Currently, HA- and GFP-tagged PLDs are being expressed in RBL-2H3 cells to determine localization and states of phosphorylation of the PLDs in unstimulated and stimulated cells. Unlike PLD1, the regulation of PLD2 is still unclear.To assess the relevance of the above findings to normal cells, we are attempting to immortalize cultured mast cells derived from human peripheral blood pluripotent stem cells by making them express constitutively active telomerase. As noted last year, the clonal expansion of CD13+ mast-cell progenitors in populations of CD34+ stem cells is preceded by transient expression of telomerase (see Z01 HL00990-13). Additional replication of mature cultured human mast cells can be induced by insertion of the telomerase gene but thus far the proliferation is not sustained. Otherwise, the mature mast cells have similar secretory and signaling responses to antigen as RBL-2H3 cells. In contrast to RBL-2H3 cells and other tumor mast-cell lines, however, the human mast cells have allowed investigation of differences in signaling processes induced by growth factors as well as antigen (see accomapnying report Z01 HL00990-14).